The study of invertebrate immunity can reveal additional basic and surprising features of innate immunity that have so far escaped our attention. As one example, a fundamental breakthrough in our understanding of the evolution of internal defense stems from the recent characterization of the immune-activating function of the dorsal/-roll pathway in Drosophila and the subsequent discovery of homologous pathways in mammals. This proposed study of the freshwater snail Biomphalaria glabrata would likely also provide novel insights regarding innate immunity. In response to infection with metazoan parasites, B. glabrata produces soluble fibrinogen-related proteins (FREPs) that consist of one or two immunoglobulin superfamily (IgSF) domains and a fibrinogen (FBG) domain. FREPs are products of a large gene family and likely mediate pathogen-recognition because they are produced by hemocytes (phagocytic defense cells) following infection, and bind parasite antigens (Adema et al, 1997). Recently, using PCR amplification from genomic DNA of B. glabrata, I have found an extraordinary diversity at both intra- and inter-individual levels within sequences of the IgSFl-encoding region of FREPs of a single subfamily (the FREP3 subfamily). More than 40 unique sequences were found in a single snail, and from several snails a total of 215 unique FREP3 sequences have been found so far. Surprisingly, Southern blotting disclosed no more than 8 FREP3-encoding loci. I hypothesize the diversity observed is most easily explained by modification of eight source sequences by gene conversion and point mutation. I further hypothesize that mechanisms exist for generation of diverse assemblages of FREPs to increase the range of antigens recognized by the innate immune system of B. glabrata. Such a mechanism may reflect the need to manage the many distinct and highly adaptable pathogens that snails routinely encounter during their lives. This hypothesis may also apply to other invertebrates. My specific aims are as follows: Specific Aim 1. Mechanism and Site of FREP3 Diversification; Specific Aim 2. Diversity of FREPs at the Expression Level; Specific Aim 3. Development of RNAi Techniques to Probe FREP Functions.